Most investigators base the description of new species of yeast on approximately 50 morphologic and physiologic criteria. This number of phenotypic traits has proven to be inadequate for the delimitation of species in yeast systematics. Nuclear DNA base composition studies have shown a wide intraspecific variation in moles per cent quanine plus cytosine in the DNA of a number of presently accepted yeast "species", suggesting that such "species" represent a conglomerate of different species that cannot be differentiated by the criteria currently used in yeast systematics. Conversely, the indiscriminate use of a limited number of such criteria for differentiation has also led to the description of many new "species", sometimes differing by only one or two biochemical abilities, such as the hydrolysis of a disaccharide. In several of the latter cases we have shown high degrees of complementarity between the DNAs from species separated on this basis. Such "species" may differ by only one or two genes or even by a single base pair in the case of point mutations. Only sparse information is available on phylogenetic relationships in yeasts. We propose the following approaches to clarify speciation in yeast and to establish phylogenetic relationships among species that appear to be related on the basis of certain phenotypic criteria. We intend: (a) to study strains of additional species (particularly strains from different ecological niches) to determine variation in nuclear DNA base composition; (b) DNA-DNA reannealing between nuclear DNAs of what appear to be closely related species with DNA base compositions that are not far apart and express the results in percent complementarity of heterologous versus homologous DNA; (c) to conduct an immunological comparison between an enzyme protein involved in the central metabolism of species shown to have significant DNA sequences in common to establish evolutionary divergence expressed in immunological distances. (d) In the case of closely related heterothallic species, interspecific cell hybridizations and degree of ascospore formation and spore viability will be correlated with the other approaches.